Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution dynamics

Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/extrusion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein we report a new method termed "Fixation-driven chemical crosslinking of exogenous ligands (FixEL)" which traps and images exogenously administered molecules-of-interest (MOI) in complex tissues. This method relies on proteins-MOI interactions, and chemical crosslinking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules and their dynamics, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOI such as PET tracer derivatives or drug-like small molecules. Clear imaging of a nanobody distributed in the whole brain was also achieved with high spatial resolution using 2D/3D mode.

Neuropathology Evaluation in Juvenile Toxicity Studies in Rodents: Comparison of Developmental Neurotoxicity Studies for Chemicals With Juvenile Animal Studies for Pediatric Pharmaceuticals

The developmental neuropathology examination in juvenile toxicity studies depends on the nature of the product candidate, its intended use, and the exposure scenario (eg, dose, duration, and route). Expectations for sampling, processing, and evaluating neural tissues differ for developmental neurotoxicity studies (DNTS) for chemicals and juvenile animal studies (JAS) for pediatric pharmaceuticals. Juvenile toxicity studies typically include macroscopic observations, brain weights, and light microscopic evaluation of routine hematoxylin and eosin (H&E)-stained sections from major neural tissues (brain, spinal cord, and sciatic nerve) as neuropathology endpoints. The DNTS is a focused evaluation of the nervous system, so the study design incorporates perfusion fixation, plastic embedding of at least one nerve, quantitative analysis of selected brain regions, and sometimes special neurohistological stains. In contrast, the JAS examines multiple systems, so neural tissues undergo conventional tissue processing (eg, immersion fixation, paraffin embedding, H&E staining only). An “expanded neurohistopathology” (or “expanded neuropathology”) approach may be performed for JAS if warranted, typically by light microscopic evaluation of more neural tissues (usually additional sections of brain, ganglia, and/or more nerves) or/and special neurohistological stains, to investigate specific questions (eg, a more detailed exploration of a potential neuroactive effect) or to fulfill regulatory requests.

FACT For Fast And Three-Dimensional Imaging of Intact Tissues

Free of Acrylamide Sodium Fast Free-of-Acrylamide Clearing Tissue (FACT) is a developed technique using no acrylamide for clearing tissues. As the lipid removal normally is a harmful process and it causes loss of biological molecules such as proteins and on the other hand is crucial for transparency and efficient antibody staining throughout the whole tissue especially for microscopy and imaging, the FACT technique is suitable since it makes chemical bonding of membrane and intracellular proteins with the extracellular matrix creating a massive three-dimensional (3D) matrix and structural support to fortify the tissue during processing. Compared to other acrylamide-based techniques, FACT requires less labor, toxic, and harmful chemicals. Here we describe protocols encompassing every angle and dimension of the FACT protocol for antibody staining and imaging of whole-cleared tissues while preserving the structure and increasing the image quality. The entire protocol includes tissue perfusion, fixation, clearing, antibody staining, Refractive Index Matching (RIM), microscopy, and imaging; this timing varies due to the size, weight, different kind of tissues and the type of immunostaining. This technique has been favorably performed on different types of tissues for molecular interrogation analysis of large tissues.

ULTRASTRUCTURAL ANALYSIS OF SCIATIC NERVE IN MICE WITH PERIPHERAL NEUROPATHY AFTER TRANSPLANTATION OF ADIPOSE-DERIVED MULTIPOTENT MESENCHYMAL STROMAL CELLS

We investigated the peripheral demyelination in transgenic mice with peripheral neuropathy and the effect of adiposederived multipotent mesenchymal stromal cells (ADSCs) transplantation on the ultrastructural features of the sciatic nerve in these mice. The B6.Cg-Tg(PMP22)C3Fbas/J transgenic mice with peripheral neuropathy were injected intramuscularly with ADSCs, which were isolated from the adipose tissue of FVB-Cg-Tg(GFPU) mice transgenic by GFP. For ultrastructural analysis, tissue fixation in animals was performed by transcardiac perfusion-fixation with 4% formaldehyde solution and 2.5% glutaraldehyde solution 16 weeks after transplantation. Electron microscopic examination of fibers of the sciatic nerve in the transgenic mice with peripheral neuropathy showed that many axons in this nerve were subjected to dys- and demyelination; the so-called onion bulb-like structures were observed. In some fibers, hypertrophy of myelin sheaths was found. In general, ultrastructural modifications in the sciatic nerve of the transgenic mice were rather similar to the pathomorphological pattern observed in patients with peripheral neuropathy. At 16 weeks after ADSC transplantation, in the sciatic nerve in mice with peripheral neuropathy thickening of the myelin sheath and increasing of the number of lamellae were observed. Thus, ADSC transplantation in mice with hereditary peripheral neuropathy has a protective effect on the ultrastructural features of the sciatic nerve and inhibits the process of axon demyelination.

Sex differences in the structure and function of rat middle cerebral arteries

Using perfusion fixation of the middle cerebral artery (MCA) in calcium-free solution at physiological pressure and systematically randomly sampling the sections prepared from the same M2 segments of MCA, we found that there are structural differences that are associated with altered cerebral blood flow (CBF) autoregulation but not neurovascular coupling and cognition in young, healthy Sprague-Dawley (SD) rats. Understanding the intrinsic differences in cerebrovascular structure and function in males and females is essential to develop new pharmaceutical treatments for cerebrovascular disease (CVD).